A multi-step reaction full-liquid-phase centrifugal microfluidic detection chip

Abstract

The application discloses a kind of to carry out multi-step reaction full liquid phase centrifugal microfluidic detection chip, including main chip, the main chip is divided into positive and negative two sides, bottom shell, preset box and reagent box, the detection chip adopts the structure of main chip, bottom shell combination, spike is set on the reverse side of main chip, bottom shell is equipped with diluent tank and reaction reagent tank, for placing preset box and multiple reagent box, can be achieved multi-step full liquid phase reaction, so that production transportation is more convenient, detection is also more convenient, and the data of simultaneous detection is also more accurate.

Description

Technical Field

[0001] This invention belongs to the field of POCT biochemical and immunoassay analysis, specifically relating to a fully liquid-phase centrifugal microfluidic detection chip that performs multi-step reactions. Background Technology

[0002] Centrifugal microfluidic chips are miniature analytical systems that integrate sample dispensing, quantification, centrifugation, reaction, and detection. Compared to traditional laboratory analysis, microfluidic integrated chips simplify the testing process. Analysts only need to add the sample to the microfluidic chip and place it in the matching point-of-care testing (POCT) instrument; no further human intervention is required, and the instrument automatically generates the corresponding test results. Therefore, operators do not need specialized experimental skills training, improving work efficiency. Because the accompanying instrument lacks conventional cleaning tubing and pipetting systems, its compact and lightweight design requires no maintenance, making it suitable for use in primary healthcare settings.

[0003] Existing centrifugal microfluidic chips, besides molecular diagnostic experiments, are mainly used for biochemical detection. Due to limitations in manufacturing processes, they are generally limited to using solid lyophilized reagents and simplified single-step reaction processes. Sample solutions and diluents are mixed using multiple slots and microchannels on the chip. The mixed liquid then flows through the microchannels into the reaction tank to react with the lyophilized reagents to obtain the desired detection results. However, this technology lacks quality control fluid calibration references, making it difficult for users to promptly identify instrument and reagent problems. When multi-step reaction detection is required, multiple reagents must be pre-placed in the same reaction chamber, turning what should be a multi-step reaction into a single step. This leads to significant deviations in the final data, rendering the final detection results unreliable. The chip is small in size, easy to carry, and convenient to operate, and can quickly obtain test results. However, it is limited by lyophilized reagents, and has problems such as complex production process, inconvenient transportation, and large deviation of test results. Therefore, it is necessary to design a detection chip that does not require lyophilized reagents. Summary of the Invention

[0004] (a) Technical problems to be solved.

[0005] To address the shortcomings of existing technologies, this invention provides a fully liquid-phase centrifugal microfluidic detection chip that performs multi-step reactions, solving the complex problems mentioned in the background art regarding the production and transportation of lyophilized reagents.

[0006] (ii) Technical solution.

[0007] To achieve the above objectives, the present invention provides the following technical solution: a multi-step reaction centrifugal microfluidic detection chip, comprising a main chip, which has two sides. The front side of the main chip includes a sample loading port, a sample separation and quantitative chamber, a diluent quantitative chamber, a mixing chamber, a reaction chamber, multiple vents, a mixing liquid arrival detection port, a sample arrival detection port, a diluent loading port, a mixing liquid quantitative chamber, a reaction reagent inlet, a colorimetric port, and multiple sets of microchannels and multiple sets of pipes for connecting and controlling fluid flow. The front side of the main chip is provided with a dense... The sealing film is characterized in that: the reverse side of the main chip includes a pre-set box puncture tip, a reagent kit puncture tip, an expansion pin, a pre-set box mounting groove (124), and a reagent kit mounting groove. The detection chip also includes a bottom shell, a pre-set box, and a reagent kit. The bottom shell includes a reagent kit lower mounting groove, a pre-set box lower mounting groove, and a pin hole. The microchannels include a first microchannel, a second microchannel, a third microchannel, a fourth microchannel, a fifth microchannel, and a sixth microchannel. The channels include a first channel, a second channel, a third channel, a fourth channel, a fifth channel, and a sixth diversion channel.

[0008] Preferably, the prefabricated box is pre-filled with diluent and has a sealing film on the upper part, which is installed between the lower mounting groove and the upper mounting groove of the prefabricated box.

[0009] Preferably, the kit contains one or more reaction reagents pre-loaded and installed between the upper and lower mounting slots of the kit. The kit has one or more puncture tips, and when there are multiple tips, there is a height difference between them. The number of puncture tips is greater than or equal to the number of reaction reagents pre-loaded in the kit.

[0010] Preferably, the sample loading port includes a sample cavity and a sample microfluidic channel, the sample cavity is connected to the sample separation and quantification cavity through a pipe, and the sample microfluidic channel is located on the side of the center of the main chip.

[0011] Preferably, the sample separation and quantification chamber includes a serum chamber and a red blood cell precipitation chamber. The serum chamber is connected to the sample chamber via a first conduit and is also connected to the sample microfluidic inlet, a first microchannel. The serum chamber and the red blood cell precipitation chamber are connected via a second conduit, with one end of the second microchannel connected to the middle of the second conduit and the other end connected to the mixing chamber. The red blood cell precipitation chamber has multiple protrusions in the middle to prevent membrane deformation that may be caused by the large span of the chamber space. The serum chamber is connected to the sample arrival detection port and the vent port via a third conduit.

[0012] Preferably, the diluent injection port is a through hole penetrating both the front and back of the main chip. It is connected to the preform box on the back of the main chip and to the diluent metering chamber on the front of the main chip through a seventh pipe. The diluent metering chamber is connected to the mixing chamber through a third microchannel. Multiple protrusions are provided in the middle to prevent film deformation that may be caused by the large span of the chamber space.

[0013] Preferably, the front side of the main chip also includes a quality control liquid sample loading port and a mixed liquid countercurrent chamber. The quality control liquid sample loading port is connected to the diluent metering chamber through a fourth pipe, and the mixed liquid countercurrent chamber is connected to the diluent metering chamber, the mixing chamber, and the venting port through a fifth pipe.

[0014] Preferably, the mixing chamber is connected to the sixth diversion pipe through the fourth microchannel, and multiple protrusions are provided in the middle to prevent the film from deforming due to the large span of the chamber space. The sixth diversion pipe is connected to the mixing liquid metering chamber and the mixing liquid arrival detection hole. The mixing liquid metering chamber is provided with multiple large metering chambers and multiple small metering chambers.

[0015] Preferably, the front side of the main chip also includes a reaction liquid countercurrent chamber, which is located between the reaction chamber and the mixed liquid metering chamber, and is connected to the mixed liquid metering chamber and the reaction chamber through a sixth microchannel. The reaction chamber is connected to the colorimetric orifice.

[0016] Preferably, the expansion pins correspond one-to-one with the pin holes, and are used to fix the main chip to the bottom shell.

[0017] Preferably, the bottom of the reagent kit lower mounting slot and the pre-made box lower mounting slot are provided with openings for lifting the reagent kit and the pre-made box.

[0018] Preferably, the main chip is fan-shaped with a central angle of 90 degrees, and a sealing film is provided on the front side. Through holes are provided at the corresponding positions of the vent, sample loading port and quality control liquid loading port.

[0019] (iii) Beneficial effects.

[0020] This invention provides a multi-step, all-liquid-phase centrifugal microfluidic detection chip, which has the following advantages: 1. The detection chip adopts a combination structure of a main chip, a bottom shell, and a packaging base. Spikes are provided on the bottom shell, and a dilution tank and a reaction reagent tank are provided. It can perform multi-step all-liquid-phase reactions, making the detection chip's detection completely consistent with the reaction system and methodology of large-scale biochemical analyzers. The reference values ​​for the project detection are also the same, making the detection results easier to accurately evaluate and verify, and making production and transportation more convenient. At the same time, the detection is also more convenient.

[0021] 2. The quality control solution sample well of the test chip directly enters the diluent quantitative chamber, reducing the flow path and allowing for standard quality control testing of the chip in the most economical way. Attached Figure Description

[0022] Figure 1 Front view of the main chip Figure 1 .

[0023] Figure 2 Front view of the main chip Figure 2 .

[0024] Figure 3 Schematic diagram of the back of the main chip.

[0025] Figure 4 Schematic diagram of the bottom shell.

[0026] Figure 5 A schematic diagram of the bottom shell, pre-set box, and reagent kit assembly.

[0027] Figure 6 Chip disassembly diagram.

[0028] Figure 7 Schematic diagram of the packaging base.

[0029] In the diagram: 1. Main chip; 101. Sample loading port; 1011. Sample chamber; 1012. Sample microfluidic channel port; 102. Sample separation and quantification chamber; 1021. Serum chamber (1021); 1022. Red blood cell precipitation chamber; 103. Diluent quantification chamber; 104. Mixing chamber; 105. Reaction chamber; 106. Exhaust port; 107. Mixture arrival detection port; 108. Sample arrival detection port; 109. Diluent loading port; 110. Mixture quantification chamber; 111. Microfluidic channel; 1111. First microfluidic channel; 1112. Second microfluidic channel; 1113. Third microfluidic channel; 1114. Fourth microfluidic channel; 1115. Fifth microfluidic channel; 112. Pipeline; 1121. First Pipeline; 1122. Second Pipeline; 1123. Third Pipeline; 1124. Fourth Pipeline; 1125. Fifth Pipeline; 1126. Sixth Split Pipeline; 113. Quality Control Solution Addition Well; 114. Mixture Backflow Chamber; 115. Reaction Solution Backflow Chamber; 116. Reaction Reagent Inlet; 117. Colorimetric Well; 121. Pre-set Box Puncture Tip; 122. Reagent Kit Puncture Tip; 123. Expansion Pin; 124. Pre-set Box Upper Mounting Slot; 125. Reagent Kit Upper Mounting Slot; 2. Bottom Shell; 21. Reagent Kit Lower Mounting Slot; 22. Pre-set Box Lower Mounting Slot; 23. Pin Hole; 3. Pre-set Box; 4. Reagent Kit. Detailed Implementation

[0030] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Example 1

[0031] Please see Figures 1-4This invention provides a multi-step reaction centrifugal microfluidic detection chip, comprising a main chip with two sides. The front side of the main chip includes a sample loading port, a sample separation and quantitative chamber, a diluent quantitative chamber, a mixing chamber, a reaction chamber, multiple vents, a mixing liquid arrival detection port, a sample arrival detection port, a diluent loading port, a mixing liquid quantitative chamber, a reaction reagent inlet, a colorimetric port, and multiple sets of microchannels and pipes for connecting and controlling fluid flow. The chip has a sealing film and is characterized in that: the reverse side of the main chip includes a pre-set box puncture tip, a reagent kit puncture tip, an expansion pin, a pre-set box mounting groove (124), and a reagent kit mounting groove. The detection chip also includes a bottom shell, a pre-set box, and a reagent kit. The bottom shell includes a reagent kit lower mounting groove, a pre-set box lower mounting groove, and a pin hole. The microchannels include a first microchannel, a second microchannel, a third microchannel, a fourth microchannel, a fifth microchannel, and a sixth microchannel. The channels include a first channel, a second channel, a third channel, a fourth channel, a fifth channel, and a sixth diversion channel.

[0032] The prefabricated box is pre-filled with diluent and has a sealing film on the top, which is installed between the lower mounting slot and the upper mounting slot of the prefabricated box.

[0033] The kit contains one or more reaction reagents pre-loaded and installed between the upper and lower mounting slots of the kit. The kit has one or more puncture tips, and when there are multiple tips, there is a height difference between them. The number of puncture tips is greater than or equal to the number of reaction reagents pre-loaded in the kit.

[0034] The sample loading port includes a sample cavity and a sample microfluidic channel. The sample cavity is connected to the sample separation and quantification cavity through a pipe, and the sample microfluidic channel is located on the side of the center of the main chip.

[0035] The sample separation and quantification chamber includes a serum chamber and a red blood cell precipitation chamber. The serum chamber is connected to the sample chamber via a first conduit and is also connected to the sample microfluidic inlet, a first microchannel. The serum chamber and the red blood cell precipitation chamber are connected via a second conduit, with one end connected to the middle of the second conduit and the other end connected to the mixing chamber. The red blood cell precipitation chamber has multiple protrusions in the middle to prevent membrane deformation that may be caused by the large span of the chamber space. The serum chamber is connected to the sample arrival detection port and the vent port via a third conduit.

[0036] The diluent injection port is a through hole that penetrates both sides of the main chip. It is connected to the preform box on the back of the main chip and to the diluent metering chamber on the front of the main chip through a seventh pipe. The diluent metering chamber is connected to the mixing chamber through a third microchannel. Multiple protrusions are provided in the middle to prevent film deformation that may be caused by the large span of the chamber space.

[0037] The front side of the main chip also includes a quality control liquid sample loading port and a mixed liquid countercurrent chamber. The quality control liquid sample loading port is connected to the diluent metering chamber through a fourth pipe. The mixed liquid countercurrent chamber is connected to the diluent metering chamber, the mixing chamber and the vent through a fifth pipe.

[0038] The mixing chamber is connected to the sixth diversion pipe through the fourth microchannel. Multiple protrusions are provided in the middle to prevent film deformation that may be caused by the large span of the chamber space. The sixth diversion pipe is connected to the mixing liquid metering chamber and the mixing liquid arrival detection hole. The mixing liquid metering chamber is provided with multiple large metering chambers and multiple small metering chambers.

[0039] The front side of the main chip also includes a reaction liquid countercurrent chamber, which is located between the reaction chamber and the mixed liquid metering chamber. The mixed liquid metering chamber and the reaction chamber are connected through a sixth microchannel, and the reaction chamber is connected to the colorimetric orifice.

[0040] The expansion pins correspond one-to-one with the pin holes and are used to fix the main chip to the bottom shell.

[0041] The bottom of the reagent kit mounting slot and the pre-made box mounting slot are provided with openings for lifting the reagent kit and the pre-made box. The number of openings at the bottom of the reagent kit mounting slot is the same as the number of reagent kits.

[0042] The main chip is fan-shaped with a central angle of 90 degrees. A sealing film is provided on the front side, and through holes are provided at the corresponding positions of the vent, sample loading port and quality control liquid loading port.

[0043] The main chip and the base shell are fixedly connected by expansion pins and pin holes. The sample is injected into the chip through the sample loading port using either injection or capillary action. The main chip and the base shell are aligned using the encapsulation base. Pressing the main chip into place causes the first reagent chamber and diluent chamber in the reagent kit to be lifted by corresponding protrusions on the encapsulation base. The spikes on the back of the main chip puncture the pre-set box film and the film on the first reaction reagent kit. The complete chip assembly, consisting of the main chip and the base shell, is loaded into the matching instrument. The instrument rotates the chip for the first time, centrifuging at a reverse speed of 4300 rpm. The sample enters the sample separation and quantification chamber through centrifugal force, and the red blood cells in the sample enter the red blood cell precipitation chamber. Serum is retained in the serum chamber, and excess serum enters the sample placement detection well through the third tube. Empty serum is discharged through the vent hole, while diluent enters the diluent quantification chamber, and reaction reagents enter the reaction chamber.

[0044] The instrument pauses for more than 30 seconds. Serum is detected in the sample placement detection well. The serum enters and fills the second microchannel through capillary action, and the diluent enters and fills the third microchannel through capillary action. The instrument drives the chip to rotate in reverse at 2500 rpm. The serum enters the mixing chamber through the centrifugal force generated by the rotation, and the diluent in the diluent metering chamber also enters the mixing chamber.

[0045] The instrument drives the chip to rotate forward and backward 10 times, so that the serum and diluent in the mixing chamber are thoroughly mixed.

[0046] If the instrument is paused for more than 30 seconds, the mixture in the mixing chamber will enter and fill the fourth microchannel through capillary action.

[0047] The instrument drives the chip to rotate at 200 rpm. The mixture enters the sixth diversion pipe through centrifugal force and then enters the reaction liquid metering chamber and the mixture position detection hole. The air in the mixture is discharged through the exhaust port.

[0048] After the mixed liquid is optically detected in the detection hole, the instrument drives the chip to rotate at more than 2000 rpm, and the quantitative mixed liquid enters each reaction chamber.

[0049] The instrument is rotated forward and backward more than 10 times to ensure that the first reaction reagent and the mixture are thoroughly mixed. The instrument then pushes the packaging base to push the reaction kit a second time, causing the film on the second reagent to be punctured.

[0050] After standing for more than 3 minutes, the first reagent reacts fully with the mixture. The instrument drives the chip to rotate at a speed of more than 2000 rpm, so that the second reagent enters the reaction chamber.

[0051] The instrument drives the chip to rotate forward and backward more than 10 times to ensure that the second reagent and the first stage reactants are fully mixed.

[0052] After standing for more than 3 minutes, the instrument is slowly rotated to record continuous readings of the colorimetric orifices connected to each reaction chamber.

[0053] A maximum of four samples can be tested in the same batch. Example 2

[0054] like Figure 1-4 Example 2 is shown. Based on Example 1, the main chip also has a quality control liquid addition port on the front. Without adding a sample, the quality control liquid addition port is directly connected to the diluent metering chamber through the fourth pipe. After mixing with the diluent, it enters the mixing chamber through the third microchannel, then enters the mixing chamber through the microchannel and the sixth diversion pipe, and finally enters the reaction chamber through the fifth microchannel. In this example, the quality control liquid directly enters the diluent metering chamber and mixes with the diluent, reducing the flow path. The quality control liquid can also be used to detect the deviation between the value read by the device and the standard value marked on the quality control liquid, and to calibrate the value read by the device. This allows for device calibration or provides a reference for subsequent sample readings, thereby obtaining more accurate detection data at the lowest cost.

[0055] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A multi-step reaction centrifugal microfluidic detection chip, comprising a main chip (1), the main chip (1) having two sides, the front side of the main chip (1) including a sample loading port (101), a sample separation and quantitative chamber (102), a diluent quantitative chamber (103), a mixing chamber (104), a reaction chamber (105), multiple venting holes (106), a mixed liquid arrival detection hole (107), a sample arrival detection hole (108), a diluent loading port (109), a mixed liquid quantitative chamber (110), a reaction reagent inlet (116), a colorimetric orifice (117), and multiple sets of microchannels (111) and multiple sets of pipes (112) for connecting and controlling fluid flow, the front side of the main chip (1) having a sealing film, characterized in that: The reverse side of the main chip (1) includes a pre-set box puncture tip (121), a reagent kit puncture tip (122), an expansion pin (123), a pre-set box mounting slot (124), and a reagent kit mounting slot (125). The detection chip also includes a bottom shell (2), a pre-set box (3), and a reagent kit (4). The bottom shell (2) includes a reagent kit lower mounting slot (21), a pre-set box lower mounting slot (22), and a pin hole (23). The microchannel (111) includes a first microchannel (1111), a second microchannel (1112), a third microchannel (1113), a fourth microchannel (1114), and a fifth microchannel (1115). The conduit (112) includes a first conduit (1121), a second conduit (1122), a third conduit (1123), a fourth conduit (1124), and a fifth conduit (1125). 25) and the sixth diversion pipe (1126) and the seventh pipe (1127), the diluent sample port (109) is a through hole that penetrates both sides of the main chip (1), and is connected to the pre-set box (3) on the back side of the main chip (1), and is connected to the diluent quantitative chamber (103) on the front side of the main chip (1) through the seventh pipe (1127). The bottom of the reagent kit lower mounting slot (21) and the pre-set box lower mounting slot (22) are provided with openings for lifting the reagent kit (4) and the pre-set box (3). The main chip (1) and the bottom shell (2) are aligned through the encapsulation base. The main chip is pressed into place, and the first reagent chamber in the reagent kit (4) and the diluent chamber in the pre-set box (3) are lifted by the corresponding protrusions on the encapsulation base. The spikes on the back of the main chip (1) pierce the film of the pre-set box (3) and the film on the first reaction reagent kit.

2. The multi-step reaction centrifugal microfluidic detection chip according to claim 1, characterized in that: The pre-installed box (3) is pre-filled with diluent and has a sealing film on the upper part, which is installed between the lower mounting groove (22) and the upper mounting groove (124) of the pre-installed box.

3. The multi-step reaction centrifugal microfluidic detection chip according to claim 1, characterized in that: The kit (4) is pre-loaded with one or more reaction reagents and installed between the upper mounting slot (125) and the lower mounting slot (21) of the kit. The kit has one or more puncture tips (122), and there is a height difference between them when there are multiple puncture tips. The number of puncture tips (122) of the kit is greater than or equal to the number of reaction reagents pre-loaded in the kit (4).

4. A multi-step reaction centrifugal microfluidic detection chip according to claim 1 or 2, characterized in that: The sample loading port (101) includes a sample cavity (1011) and a sample microfluidic port (1012). The sample cavity (1011) is connected to the sample separation and quantification cavity (102) through a pipe (112). The sample microfluidic port (1012) is located on the side of the center of the main chip (1).

5. A multi-step reaction fully liquid-phase centrifugal microfluidic detection chip according to claim 4, characterized in that: The sample separation and quantification chamber (102) includes a serum chamber (1021) and a red blood cell precipitation chamber (1022). The serum chamber (1021) is connected to the sample chamber (1011) through a first conduit (1121). The serum chamber (1021) is connected to the first microchannel (1111) of the sample microchannel opening (1012). The serum chamber (1021) is connected to the red blood cell precipitation chamber (1022) through a second conduit (1122). One end of the second microchannel (1112) is connected to the middle of the second conduit (1122), and the other end is connected to the mixing chamber (104). The red blood cell precipitation chamber (1022) has multiple protrusions in the middle to prevent the membrane from deforming due to the large span of the chamber space. The serum chamber (1021) is connected to the sample arrival detection hole (108) and the exhaust hole (106) through a third conduit (1123).

6. The multi-step reaction centrifugal microfluidic detection chip according to claim 1, characterized in that: The diluent metering chamber (103) is connected to the mixing chamber (104) through the third microchannel (1113), and multiple protrusions are provided in the middle to prevent the film from deforming due to the large span of the chamber space.

7. The multi-step reaction centrifugal microfluidic detection chip according to claim 1, characterized in that: The front side of the main chip (1) also includes a quality control liquid sample loading port (113) and a mixed liquid countercurrent chamber (114). The quality control liquid sample loading port (113) is connected to the diluent metering chamber (103) through a fourth pipe (1124). The mixed liquid countercurrent chamber (114) is connected to the diluent metering chamber (103), the mixing chamber (104) and the vent (106) through a fifth pipe (1125).

8. The multi-step reaction centrifugal microfluidic detection chip according to claim 1, characterized in that: The mixing chamber (104) is connected to the sixth diversion pipe (1126) through the fourth microchannel (1114). Multiple protrusions are provided in the middle to prevent the film from deforming due to the large span of the chamber space. The sixth diversion pipe (1126) is connected to the mixing liquid metering chamber (110) and the mixing liquid arrival detection hole (107). The mixing liquid metering chamber (110) is provided with multiple large metering chambers and multiple small metering chambers.

9. A multi-step reaction centrifugal microfluidic detection chip according to claim 1, characterized in that: The front side of the main chip (1) also includes a reaction liquid countercurrent cavity (115), which is located between the reaction cavity (105) and the mixed liquid metering cavity (110). The mixed liquid metering cavity (110) and the reaction cavity (105) are connected through the fifth microchannel (1115). The reaction cavity (105) is connected to the colorimetric orifice (117).

10. A multi-step reaction centrifugal microfluidic detection chip according to claim 1, characterized in that: The expansion pins (123) correspond one-to-one with the pin holes (23) and are used to fix the main chip (1) to the bottom shell (2).

11. A multi-step reaction fully liquid-phase centrifugal microfluidic detection chip according to claim 7, characterized in that: The main chip (1) is fan-shaped with a central angle of 90 degrees. A sealing film is provided on the front side. Through holes are provided at the corresponding positions of the exhaust hole (106), sample loading port (101) and quality control liquid loading port (113).

Images